This invention relates to a novel polypeptide having acid residues derived from a strong acid and a process for measurement of analytes to be measured in samples derived from living bodies, such as body fluids (e.g. serum, blood, plasma, urine, etc.), lymphocyte, hemocyte, and various cells, with the use of said polypeptide.
It is known that specific substances interact strongly on each other (namely, they have a high affinity for each other) to form a stable complex. The specific substances include, for example, the following combinations: antigen and antibody; protease and its inhibitor; sugar chain and lectin; enzyme and substrate therefor or coenzyme; physiologically active substance such as hormone, and receptor or transport protein for said active substance; and a pair of polynucleotide chains of duplex DNA.
As a method for measuring an analyte to be measured in a sample by utilizing the above interaction, the following can be exemplified: a method of forming a complex on a solid phase by the reaction of substances in the above-exemplified combination and then carrying out so-called Bound/Free (B/F) separation by use of said solid phase such as enzyme immunoassay (EIA), radioimmunoassay (RIA) and fluoroimmunoassay (FIA) (for instance, the processes disclosed in JP-A 1-22706 (EP-A-326100), JP-A 3-44399, etc.); and a method developed by some of the present inventors, e.g., a method of carrying out separation of a complex from a free analyte to be measured, i.e. the so-called B/F separation, by use of a high-pressure liquid chromatography (HPLC) (for example, the processes disclosed in JP-A 2-28557 (EP 357869), JP-A 3-206964, JP-A 3-221865, JP-A 6-66800, etc.).
In such a measuring method, the precision of measurement and the time required for analysis are influenced by the efficiency of the B/F separation to a certain extent. Therefore, various researches on the B/F separation have been conducted.
For example, in the processes disclosed in JP-A 1-227061 (EP-A 326100), JP-A 3-44399, etc., in which the B/F separation is carried out using a solid phase, and the processes disclosed in JP-A 6-66800, etc., in which the B/F separation is carried out using HPLC, the B/F separation is carried out, for example, as follows: an anionic substance is previously introduced into, for instance, an antibody to an analyte to be measured in a sample derived from a living body, and the so-called B/F separation is carried out by utilizing anionic properties of the anionic substance in a complex of the analyte and the antibody.
Such a method makes it possible to carry out the B/F separation more efficiently than before. However, in all the processes, the anionic substance used is that derived from a poly(amino acid), and the B/F separation is carried out by utilizing the anionic properties imparted by the carboxyl groups of the poly(amino acid), and thus there are found such drawbacks as mentioned below in case where the B/F separation is carried out using HPLC.
Namely, in the method using HPLC, the complex should be separated from, for example, free antibody and substances present in the sample which tend to affect the measurement, simultaneously with the B/F separation. For carrying out such separation sufficiently by utilizing the anionic properties of carboxyl groups derived from the poly(amino acid), at least about 200 carboxyl groups (about 200 amino acid residues) should be introduced into the anionic substance molecule. Accordingly, there have been the following problems. First, the preparation of the anionic substance requires much labor. Moreover, the large-scale preparation of the anionic substance with a uniform molecular weight is difficult, so that when the B/F separation is carried out by HPLC by utilizing such an anionic substance, tailing or leading of a peak occurs to lower the precision of measurement in some cases. In addition, when a poly(amino acid) having carboxyl groups, such as poly(glutamic acid) is used for the separation, a nonspecific reaction takes place to cause a phenomenon such as an increase in a blank value and measured values in some cases. For preventing the phenomenon, it is necessary to add a suitable anionic polymer such as xcex3-poly(glutamic acid).
Thus, there is a desire to seek further improvement.
The present invention was made in view of such conditions and is intended to provide a novel polypeptide and a process for measuring an analyte to be measured in a sample derived from a living body which uses the polypeptide. When a complex formed by the interaction between an analyte to be measured in a sample derived from a living body and a substance having affinity for the analyte (hereinafter abbreviated as xe2x80x9caffinity substancexe2x80x9d) is separated from free affinity substance, i.e. non-reacted affinity substance, and substances present in the sample which tend to affect the detection of the complex, by a method applying negative charge, e.g. a method using an anion-exchanger, said polypeptide can be used for separating the complex from the free affinity substance and the like more effectively.
The present invention provides a polypeptide having at least three acid residues derived from a strong acid.
The present invention also provides a combined product of the polypeptide and an affinity substance for an analyte to be measured in a sample derived from a living body.
The present invention further provides a compound, in which a maleimido group is bound through a spacer to the N-terminus of the polypeptide, and a combined product of said compound and a substance having a SH group and affinity for an analyte to be measured in a sample derived from a living body.
The present invention still further provides a reagent for measuring an analyte to be measured in a sample derived from a living body, which comprises a combined product of the polypeptide and an affinity substance for the analyte, or the combined product mentioned above having a maleimido group-spacer-polypeptide bonding.
The present invention still further provides a process for measuring a living body component which comprises reacting a sample derived from a living body with a reagent comprising a combined product of the polypeptide and an affinity substance for an analyte to be measured in the sample or a combined product of the compound having a maleimido group-spacer-polypeptide bonding and an affinity substance having a SH group and affinity for an analyte; separating the resulting complex; and determining the amount of the living body component in the sample on the basis of the amount of the complex.